Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site.
Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-alpha-D-galactopyranosylpeptide galactose beta(1 lead to 3) transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements. Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.
D000116:Acetylgalactosamine / Q000494:pharmacology; D001665:Binding Sites; D002413:Cations, Divalent; D003902:Detergents / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D005700:Galactosyltransferases / Q000097:blood*; D006801:Humans; D007700:Kinetics; D009994:Osmolar Concentration; D013696:Temperature
D002413:Cations, Divalent; D003902:Detergents; D005700:Galactosyltransferases; D000116:Acetylgalactosamine